Eosinophils, basophils and myeloid-derived suppressor cells in chronic Loa loa infection and its treatment in an endemic setting

Background Chronic infection by Loa loa remains an unsolved immunological paradox. Despite harboring subcutaneously migrating adult worms and often high densities of microfilariae, most patients experience only relatively mild symptoms, yet microfilaricidal treatment can trigger life-threatening inflammation. Here, we investigated innate cell populations hypothesized to play a role in these two faces of the disease, in an endemic population in Gabon. Methodology/Principal findings We analyzed numbers and activation of eosinophils and basophils, as well as myeloid-derived suppressor cell (MDSC) subsets and associated circulating cytokine levels by flow cytometry in sex- and age-matched L. loa-uninfected (LL-), -amicrofilaraemic (MF-) and -microfilaraemic (MF+) individuals (n = 42), as well as microfilaraemic individuals treated with albendazole (n = 26). The percentage of eosinophils was lower in LL- (3.0%) than in the combined L. loa-infected population, but was similar in MF+ (13.1%) and MF- (12.3%). Upon treatment of MF+, eosinophilia increased from day 0 (17.2%) to day 14 (24.8%) and had decreased below baseline at day 168 (6.3%). Expression of the eosinophil activation marker CD123 followed the same pattern as the percentage of eosinophils, while the inverse was observed for CD193 and to some extent CD125. Circulating IL-5 levels after treatment followed the same pattern as eosinophil dynamics. Basophil numbers did not differ between infection states but increased after treatment of MF+. We did not observe differences in MDSC numbers between infection states or upon treatment. Conclusions/Significance We demonstrate that both chronic infection and treatment of L. loa microfilaraemia are associated with eosinophil circulation and distinct phenotypical activation markers that might contribute to inflammatory pathways in this setting. In this first ever investigation into MDSC in L. loa infection, we found no evidence for their increased presence in chronic loiasis, suggesting that immunomodulation by L. loa is induced through other pathways.

Results of baseline stool microscopy in 18 out of 26 participants included in the immunological analysis of the treatment study, for whom these results were available.and at the end of follow-up (D168) (right column, Tukey's boxplot gives median, interquartile range (IQR) and whiskers 1.5 IQR).Blue dashed lines represent lower limits of quantification (1 pg/ml).P values were obtained by Friedman's and Nemenyi's post-hoc test.Matched cytokine data were unavailable for nine participants in the cross-sectional and five participants in the treatment study.

Figure B :
Figure B: Relative microfilaraemia of participants in the clinical trial Relative microfilaraemia (normalized to mean of screening and baseline) of 26 microfilaraemic participants undergoing albendazole-based treatment regimens (3 weeks of albendazole, 3 + 2 weeks of albendazole or 3 weeks albendazole + single-dose ivermectin).Grey lines individual data, black dots the median, the dotted line the baseline microfilaraemia and the arrows sampling for immunology.

Figure C :
Figure C: Correlation of eosinophil activation and baseline microfilaraemia Correlation of percentage of SSC hi CD125+ CD193+ eosinophils among single leukocytes and expression of surface activation markers as median fluorescence intensity (MFI) or percentage positive and baseline Loa loa-microfilaraemia in the cross-sectional and treatment study.Black lines and shaded areas represent linear regression lines with 95 % confidence bands.R and p values were obtained by Spearman correlation analysis.

Figure D :
Figure D: Correlation of eosinophil activation and drop in microfilaraemia upon treatment Correlation of day 0 to day 14 fold change of percentage of SSC hi CD125+ CD193+ eosinophils among single leukocytes and expression of surface activation markers as median fluorescence intensity (MFI) or percentage positive and day 0 to day 14 drop in Loa loa-microfilaraemia as percentage of day 0 microfilaraemia in 20 individuals undergoing treatment with albendazole.Black lines and shaded areas represent linear regression lines with 95 % confidence bands.R and p values were obtained by Spearman correlation analysis.

Figure E :
Figure E: T cell proliferation-suppression assayProliferation indices of CD4+ and CD8+ single live CFSE-stained healthy donor peripheral blood mononuclear cells (PBMC) after undergoing a four-day proliferation-suppression assay with increasing ratios of co-cultured polymorphonuclear myeloid-derived suppressor cells (MDSC, blue) or leukocyte controls (PMN, grey) from five study participants.Proliferation indices were normalized to the stimulated control (healthy donor PBMC only).Dots and error bars represent mean and standard error of the mean, respectively.One-sided p values were obtained by paired Wilcoxon signed-rank tests.

Table C : Immunological outcomes by gastrointestinal helminth co-infection No GI helminth co-infection GI helminth co-infection
Main baseline immunological outcomes by gastrointestinal helminth co-infection in 18 out of 26 participants included in the immunological analysis of the treatment study, for whom these results were available.Gastrointestinal co-infection was defined as detection of ova of Trichuris trichiura, Ascaris lumbricoides and/or hookworm spp.and/or Strongyloides stercoralis larvae by microscopy in a participant's stool.